[8]
E. Uchimura, H. Machida, N. Kotobuki, M. Ikeuchi, M. Hirose, J. Miyake and H. Ohgushi, submitted. Acknowledgments This work was done by Three-Dimensional Tissue Module Project, METI (A Millennium Project) and in part supported by the R&D Projects in Advanced Support System for Endoscopic and Other Minimally Invasive Surgery, entrusted from the New Energy and Industrial Technology Development Organization (NEDO) to the Japan Fine Ceramics Center.

Abstract: We have cultured mesenchymal cells (MSC) on various types of ceramic disks and used these tissue-engineered ceramics for hard tissue regeneration. In this approach, observation of cultured cell morphology is important even if culture substrata are calcium phosphate ceramics, which usually show bioactive nature. However, due to the opaque nature of the ceramics, cells observation is very difficult. Here, we demonstrate light microscopic observation of rat MSC cultured
on transparent β-tricalcium phosphate ceramics (β-TCP). The culture was performed in osteogenic medium. Thus, the cell differentiated into bone-forming osteoblasts, which fabricated a mineralized matrix on the ceramic disks. Microscopic observation revealed that the cascade of osteogenic differentiation after attachment/proliferation of MSC on the ceramic disks was similar to that on a
culture grade polystyrene dish. These results confirmed the excellent property of β-TCP for MSC culture leading to hard tissue regeneration.

Abstract: Fibronectin (FN) and type I collagen (Col), which are kinds of extracelluar matrices, were coprecipitated with calcium phosphate to form a composite layer on a hydroxyapatite (HAP) ceramic using a supersaturated calcium phosphate solution (CP solution). The amounts of protein immobilized in the layers were determined to be 20.97±3.04 µg·cm-2 for FN, 5.26±0.19 µg·cm-2 for Col and 21.72±2.30 µg·cm-2 for simultaneously immobilized FN and Col. When osteoblastic
MC3T3-E1 cells were cultured on the HAP ceramics with the composite layer containing FN and/or Col, calcified tissue was formed through the activity of the cells. The result showed that the composite layer accelerated the differentiation of MC3T3-E1 to bone-forming cells. It is assumed that osteoblastic cells in alveolar bone migrated and differentiated on the surface of the tooth roots
when the artificial tooth roots were covered with the composite layer.

Abstract: Similar to other glucocorticoids, dexamethasone (DEX) induces osteoblast differentiation. At high concentrations, glucocorticoids may induce osteoporosis as a side effect. However, the exact mechanism of these two opposing effects has not been elucidated. To understand the mechanism of DEX-induced osteoblast differentiation, we developed a real-time osteoblast differentiation detection system using dual labeling of cells with fluorescent proteins. The promoter sequences of type I collagen and osteocalcin were ligated with mCherry and green fluorescent protein (GFP), respectively. Type I collagen is an early marker of osteoblast differentiation, and osteocalcin is a terminal differentiation marker. We investigated the effects of DEX on cell proliferation and differentiation using cells transformed with both constructs. Low DEX concentrations (<10 μM) induced calcification, as determined by alizarin-red staining, whereas calcification was inhibited at higher concentrations (>100 μM). Consistent with these results, mCherry-associated red fluorescence as an early marker was evident under both conditions, whereas green fluorescence associated with terminal differentiation was evident only at lower DEX concentrations. The level of green fluorescence diminished in a DEX-concentration-dependent manner. Thus, DEX does not inhibit the early stages of osteoblast differentiation but instead inhibits terminal differentiation.